developing real world tests and assays using gold particles arista biologicals inc

Developing Real World Tests and Assays Using Gold Particles

Arista Biologicals Inc.

Developing Real World Tests and Assays

1. Biological Reagent Concerns

2. Assay Characteristics

3. Conjugate Optimization

4. Membrane Optimization

5. Conjugate Pads

6. Sample Pads

7. Problem Solving

8. Clinical and Stability Testing


Biological Reagent Concerns

Monoclonal antibodies Most reproducible - True since no change in affinity or specificity

theoretically Most sensitive and specific - Chosen for best optimal behavior Easiest to conjugate - Since you are dealing with a homogeneous

protein

Polyclonal antibodies Fastest to produce - Good response in 45-90 days Generally least expensive - Unless affinity purified Least reproducible - Variation from animal to animal and over time in

the same animal Most cross-reactivity - Usually not desirable except for looking for

variants etc.


Biological Reagent Concerns Cont…

Antigens Hapten - Carrier conjugates, small molecule assays Recombinant antigens - Very reproducible but may not cover variants and do not

always fold as native protein Native antigens - Some variation from lot to lot and usually a mixture of proteins.

In many cases less sensitive assay is produced. Many times are denatured Synthetic peptides - Inexpensive but less like native forms. Usually need to be

conjugated to a carrier

Matching Compatibilities Cross-reactivity - Epitope overlapping, not enough specificity in the reagents,

too much specificity in the reagents

Interspecies interactions - Usually observed in assay systems containing

antibodies from different species

Prozoning - Usually observed with non affinity purified antibodies or assays

requiring extremely high levels of antigen detection



Purity Methods - Protein A and G, ion exchange, affinity

purifications, and other chromatographic methods Effects on producing conjugate - Irreproducible

sensitivities, aggregation of some conjugates etc. Effects on specificity - Background reactions sometimes

immunological in nature and sometimes protein-protein interactions are observed.

Effects on sensitivity - Less sensitive due to less binding sites available

Testing - Generally electophoresis and chromatographic methods acceptable. HPLC especially useful looking for aggregation of the proteins



Buffers Compatibility with conjugation method - Several

types of buffers promote aggregation of the

conjugate during formation. Many conjugate to the

probe directly.

Compatibility with membrane coating - Many

buffers contain salts or other materials which

compete for binding sites with the membrane



Concentrations Solubility Issues - Typically problems with

recombinant, native, and synthetic peptides because

of hydrophobic characteristics

Typical Ranges 1-4 mg/ml for antibodies

1 mg/ml for protein antigens

4mg/ml do to ug/ml for Haptens


Assay Characteristics

Format - Flow through or lateral flow (dipstick, device, or midstream). May require

specific components including pads, membranes, pretreatments

Serum, Urine, Whole Blood, Feces, Dirt, Air, etc. - Test may be

desired to work with several for the same assay.

Dilution of sample - Creates another step but allows standardization of the

sample

Sensitivity - May require large amounts of reagents or slower assay development

times

Time of assay - If your requirements are for a faster assay, larger

amounts of reagents may be required

Cost - Limitations on the amounts of reagents, type of components, etc.


Conjugate Optimization

Type of Conjugate Latex or colloidal gold - Color desired, type of assay,

sensitivity, amounts of reagents required, etc. Covalent or passive absorption

Size of Particle Type of particle Sensitivity desired - Larger particles generally give stronger

signals but more nonspecific interaction problems. Conversely, smaller particles may be used to cure a non-specific binding problem

Cost of protein to be conjugated


Conjugate Optimization Cont…

Sensitivity Desired Coating levels - Passive absorption vs. covalent

attachment issues

Method to Immobilize Conjugate Dispensing - Can use high concentration with low

volumes. Higher conjugation ratios may also be

required

Soaking - May require vacuum drying or lyophilization

depending on how critical conjugate quantity per test is

Arista Biologicals Inc

Membrane Optimization Quality and Cost

Quality and cost vary from manufacturer to manufacturer and from material to material.

Concentration Ranges Typically in sandwich assays 1-4 mg/ml, protein antigens

usually range from 0.1-1.0 mg/ml, and Hapten assays may require 1 microgram to 4 mg/ml.

Sensitivity Desired A higher flow rate membrane will generally develop faster

but require more reagents to obtain the same sensitivity. Smaller pore size membranes also bind more capture protein

.Arista Biologicals Inc.

Membrane Optimization Cont…

Flow Rate Generally the higher the flow rate required the larger the

pore size needed. Larger pore sizes generally have less chance of aggregation of the reagents causing flow problems

Blocking or Not Blocking Blocking takes care of any nonspecific binding and can be

used to change flow rates and other characteristics of the native membrane. It is also time consuming in many cases. Several blocking agents may be required to be tested including BSA, PEG, PVA, PVP, etc.


Conjugate Pad Optimization

Considerations Polyester, rayon, glass fiber materials most widely used Most of these materials are hydrophobic naturally and need to

be treated during or prior to conjugate immobilization Flow rates may vary by the material or the treatment Treatment usually includes addition of polymer and surfactant Sugars may or may not be required in the conjugate upon

immobilization depending on the method Conjugate can be dispensed up to approximately 3 to 4

microliters per cm. Dispensing is much more accurate than soaking methods


Conjugate Pad Optimization Cont…

Considerations Cont… Treatments can be varied to make the product more or

less sensitive to humidity and temperature conditions

Conjugate can be formulated to vary the rate of release

of the conjugate during actual assay. This is sometimes

key in determining sensitivity. It also can save on reagent

Blocking reagents may be included in this pad if

membrane is not blocked

Assay buffer may be included in the pad if compatible

with conjugate stability and resolubilization.


Sample Pad Optimization

Is a Sample Pad Required? If the conjugate is stable and resoluble in

the assay buffer a one bottom pad system

can be utilized. The material must also have

acceptable flow and liquid capacity

characteristics


Sample Pad Optimization Cont…

Material Requirements Flow characteristics - Does it flow at the proper rate in order not to

inhibit the rate of assay development

Volume capacity - Does it hold enough buffer for the components to

be in high enough amounts to have the proper effect for the entire

range of clinical samples

Nonspecific binding of antigen - Does the analyte interact with the

material nonspecifically

Ease of handling - Can the material be processed with out

damaging it during the normal manufacturing conditions


Sample Pad Optimization Cont… Buffer Components

pH buffering capacity - the pad must control the pH of the assay during use.

Ionic strength control - the pad must contain proper amounts of salts to control the ionic strength of the assay

The pad material - must be blocked of any nonspecific binding of the analyte if the material does not already have that characteristic

The pad material- must contain any surfactants required for specificity requirements of the assay. It must reduce these type of nonspecific interactions

The pad material - must contain any polymers or protein needed to block either immunological reactions from occurring nonspecifically or protein-protein interactions not desired

Note: If membrane blocking reagent is required the sample pad normally will contain this reagent and/or the conjugate pad


Typical Problem Observations and Solving

Nonspecific Binding Heterophile Reactions

absorption addition of IgG addition of surfactants antibody fragmentation pH changes addition of “hard” buffers


Typical Problem Observations and Solving Cont…

Nonspecific Binding Cont… General Nonspecific Reactions

blocking changing blocking agent on membrane change blocking agent on conjugate surfactant addition smaller particle production pH changes buffer changes decreasing reagents or strength change in conjugation procedure (method)



Lack of Sensitivity Increase capture material Increase conjugate Increase strength of conjugate Increase particle size Decrease or change blocking agent Decrease flow rate

decrease pore size increase blocking agent

Decrease rate of conjugate release Use higher affinity antibodies or stronger antigens Increase activity of present reagents through repurification Add activity enhancer - such as PEG Decrease or change surfactants Check for analyte absorption onto assay component



Large Volumes of Reagent Required Increase particle size Decrease flow rate Increase activity or purity of reagent Decrease rate of conjugate release Dispense conjugate

Lack of Specificity (Immunological) Repurify (affinity chromatography, absorption) Scavenger antibodies and antigens Find new reagents



Migration Problems Remake conjugate

smaller particle different particle change protocol for production (chemistry changes) change or increase blocking agent add surfactants block membrane directly look for leaching of reagents from one material to

another



Stability Problems Change conjugate pad material Change preparation of conjugate pad

increase or add surfactants increase or add polymers change pH or buffer add or increase sugars to conjugate change sugars in conjugate

Look for moisture problems Change membrane material Add preservatives to membrane block Add preservatives to capture protein prior to dispensing Look for leaching of reagents from one material to another Look for degradation of materials on sample pad


Clinical and Stability Testing Clinical Testing

Highly probable problem samples should be tested first. Suspected crossreactant containing samples, heterophile type samples, low sensitivity samples, variants, lipemic, hemolyzed, etc. samples should be tested early before large complete final clinical tests are performed. Reformulation should be performed as required

If new formulations or materials are used than before especially new blocking formulations, conjugate pad formulations, membranes, etc. then some accelerated stability studies should be performed. Both biological stability and changes in physical characteristics such as flow should be observed closely

During clinical testing several technicians should be used especially when performing in house testing looking for slight variations in users techniques

Enough clinical testing should be performed using challenging samples which would be expected to be encountered in the field


Clinical Testing and Stability Cont…

Stability Proper stability testing should be carried out. If real time stability cannot be

used the product should be tested at several different temperatures. At least

three different temperatures is recommended. These assays are

multicomponent biological systems and cannot be treated a simple chemical

reactions. However, we have observed using identical formulations and

procedures for key components as a rule do follow a pattern

We have also found that fresh product sometimes does not perform the

same as slightly aged product. Several drying or curing steps seem to take a

few days to complete. In some cases performance increases. This is

especially important when producing assays which require high precision

optimization for every batch such as DOA tests


developing real world tests and assays using gold particles arista biologicals inc

Documents

biological reagent concerns2

haptensarista biologicals

proteinsarista biologicals

membranearista biologicals

sensitive assay

conjugate optimization4

conjugate pads6

proteinprotein interactions